ABSTRACT Sepsis is a multi-organ, dysfunctional response to infection. PROJECT 4 focuses on the role of released neutrophil extracellular traps (NETs), its ability to entrap microbes, and NET-degradation products (NDPs, e.g., cell-free DNA and histones) in organ damage, and on the interactions of the cationic chemokine, platelet factor 4 (PF4) and the polyanion heparin with NETs. On presentation, many septic patients have an overwhelming amount of released NETs so that preventing NET release (NETosis) by blocking peptidylarginine deiminase 4 or accelerating NET lysis would be ineffective in preventing morbidity and mortality. We propose that in sepsis NET stabilization, enhanced microbe entrapment, and/or NDP sequestration would be protective. Our studies have defined three related strategies that can achieve one or more of these goals. We propose to better understand the underlying mechanism(s) of these strategies, hemostatic/thrombotic implications, and their potential therapeutic efficacy. Specific Aim (SA) #1: Understanding small, positive protein effects on NETs. PF4 and other small, positive proteins (e.g., protamine sulfate (ProSO4)) compact NETs, decreasing their lysis by DNases. PF4, but not ProSO4, prevents NDP release, an important difference between these cationic proteins that will be explored to understand the impact of compacting NETs in sepsis. PF4 enhances microbe entrapment and protects the endothelium from NET injury. Whether ProSO4 similarly effects NET biology given that it releases NDPs will be pursued in a microfluidic system and in murine endotoxic/sepsis models. Both cationic proteins will be compared to or in conjunction with other NET-directed therapeutic options. The side-effects of these cations, especially on hemostasis, will be explored. Pilot trials of canine and human (h) PF4s will be infused in dogs with spontaneous peritonitis with the long-term goal of a clinical trial in this large animal sepsis setting. SA#2: Studies on enhancing the effect of PF4 on NETs. We have found that a monoclonal antibody KKO that enhances PF4 binding to NETs further increases DNase resistance. An Fc-modified KKO variant protects against NET deleterious effects in vitro and in mice models of sepsis. The underlying mechanism(s) of how KKO, and other anti-PF4 antibodies effect NET biology will be defined. KKO infusion studies will be pursued in mice expressing hPF4 looking at efficacy and thrombotic complications and in pilot studies of co-infused KKO and hPF4 in septic dogs. SA#3: Understanding ODSH effects on NETs in sepsis. We have previously shown that the desulfated heparin ODSH, which has markedly lower anticoagulant effects than heparin, is protective in a histone infusion murine model and will explore its mechanistic basis in vitro and in murine sepsis models as in SA#1. We will continue a dose escalation study on the efficacy of ODSH in dogs with spontaneous peritonitis to define maximal tolerated dose for an eventual clinical trial. These studies in PROJECT 4 should advance our understanding of the interactions of PF4 and heparins with NETs in sepsis and lead to novel therapies for this devastating clinical state.